Although there has been extensive work developing microfabrication and microfluidic technology for biomedical applications, bottlenecks remain in moving the technology broadly into biomedical research laboratories. Part of the issue is a lack of familiarity with the capabilities of microfabrication on the part of many biomedical researchers. In addition, many potential research projects have constraints that require extensive customization and multiple design iterations, which may not be achievable with the limited number of commercial products available. In an effort to lower the barriers for applying microfabrication techniques to a wide range of biomedical problems, we have developed an in-house microfabrication capability for making templates for PDMS or hydrogel devices using either a dry-film resist or SU-8. Although the resolution, device yield, and complexity are somewhat lower than those achievable with a dedicated cleanroom, they are nonetheless sufficient for many experiments on cells. Furthermore, the instrumentation complexity, fabrication cost, and turnaround time are greatly reduced, enabling rapid cycling through design parameters as needed. This year, we continued to refine protocols for patterning and device fabrication, and are now able to reliably pattern single and double layer template features with lateral dimensions under 5 microns, and with heights ranging from a few microns to a few hundred microns. The ability to pattern multiple layers with different heights on a single template enables the production of devices with substantially greater functionality as an example, structures that can be used for trapping cells in one compartment while providing continuous fluid delivery through larger channels. We have also developed protocols for using these templates to generate microstructured PDMS, agarose, and PEGDA hydrogels, and this year have been refining techniques for making thin (<200 micrometer) PDMS layers for use in multilayer devices and bottomless structures. We continue to develop protocols for surface modification of PDMS and other polymers, including the irreversible bonding of PDMS to glass, using instrumentation in our laboratory, as well as techniques for connecting devices to flow-control instruments, such as syringe pumps and pressure controllers. Through continued collaboration with scientists at NIST, we are also able to access the nanofabrication facilities at NIST to make more complex and higher-tolerance structures as needed. This year, we also developed the use of a programmable razor cutter and pressure-sensitive adhesive to directly make flow cells with heights ranging from 25 to a few hundred microns, and sub-millimeter lateral dimensions. This low-cost and convenient method is useful for ready fabrication of flow cells with two glass walls, as well as for providing fluidic confinement over already-functionalized surfaces. These capabilities have found application in a number of projects, representing a broad variety of interests and institutes, over this past year. In addition to the representative projects discussed below and others still in the early stages, we have also trained researchers in basic microfabrication techniques, including personnel from other laboratories in NHLBI, NCI, NICHD, and NIBIB. One longer-running project is an ongoing effort in collaboration with LSB, NIAID, to study chemotaxis in 3-D collagen matrices, for which we have been developing and refining a microfluidic agarose device compatible with high-resolution fluorescence and two-photon imaging. A mixing tee on a separate platform, together with programmable syringe pump flow control, enables the formation of reproducible time-varying spatial gradients. This year, we have continued to explore different architectures for a multilayer device capable of independently controlling the degree of collagen fiber alignment and the chemical gradient. In addition, we have developed finite element models of the original device as well as the multilayer device in order to inform device design and assist in characterization. A second project is the continuing development, in collaboration with LCE, NHLBI, of gratings for phase-contrast x-ray imaging, which involves the use of the NIST nanofabrication facilities as well as our own equipment. This year, our group has contributed to refinement of the grating fabrication parameters and characterization as well as to the development of protocols for electrodeposition of metals into the patterned grating structures. A third ongoing project, in collaboration with MDP, NIDDK, is the use of microwells to confine eggs in order to study fertilization with fast, high-resolution microscopy. For this application, the wells need to be compatible with existing fluorescence imaging and culture systems, and also to enable capture of the eggs with minimal handling. A recent refinement has added microchannels to enable controlled delivery of the sperm at the focal plane. In a fourth project we have been continuing the development, fabrication, and characterization of thin hybrid polymer films made by spin coating for use in operator-independent, high-resolution, laser capture microdissection. This work, begun as part of an inter-institute Director's Challenge project with researchers in NIMH, NCI, and NICHD, is aimed at developing methods for tissue-based capture of subcellular structures for mass spectrometry-based proteomic analysis. A fifth project is the development and implementation of a PDMS microfluidic gradient generator for the deposition of chondroitin sulfate proteoglycans on substrates for neural cell culture, that we are using, in collaboration with DN, CBPC, NHLBI, to gain better understanding of the role these molecules play in axon growth and guidance. Finally, we are continuing work on a project aimed at miniaturizing the luciferase immunoprecipitation system (LIPS) assay developed in LSB, NIDCR, which uses a fusion protein consisting of Renilla luciferase and an antigen of interest to probe for antibodies in human serum. This year, we demonstrated successful function of the assay in a glass flow cell using power-free flow control, and achieved more than a thirty-fold increase in signal over the original, single-channel microfluidic format by using functionalized magnetic beads to increase the effective surface area for capture. In addition, we began work on a battery powered detector using a large-area photodiode. Our ongoing work is focused on further boosting the signal, and on incorporating passive flow controls into the device design in order to improve ease of use.